Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and discuss potential roles in template-mediated non-enzymatic polymerization of RNA and other related biomolecules, functions of ribozymes and aptamers, and benefits or penalties imparted by liquid demixing. We conclude that tiny liquid droplets may have concentrated precious biomolecules and acted as bioreactors in the RNA World.

Owing to their ability to encapsulate biomolecules, complex coacervates formed by associative phase separation of oppositely charged polyelectrolytes have been postulated as prebiotic nonmembranous compartments (NMCs). Recent studies show that NMCs sequester RNA and enhance ribozyme reactions, a critical tenet of the RNA World Hypothesis. As RNA is negatively charged, it is expected to interact with polycationic coacervate components. The molecular basis for how identity and concentration of polyanionic components of complex coacervates affect ribozyme catalysis remains unexplored. We report here a general mechanism wherein diverse polyanions enhance ribozyme catalysis in complex coacervates. By competing for unproductive RNA-polycation interactions, polyanions enhance ribozyme reaction more than 12-fold. The generality of our findings is supported by similar behavior in three polyanionspolycarboxylates, polysulfates, and polysulfates/carboxylatesas well as two different ribozymes, the hammerhead and hairpin. These results reveal potential roles for polyanions in prebiotic chemistry and extant biology.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable in vitro, these structures remain difficult to study in vivo. One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

Intracellular condensates formed through liquid–liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.